IgM-MEDIATED RECEPTOR CLUSTERING AND CELL MODULATION

ABSTRACT

Materials and methods for using multivalent molecules (e.g., antibodies) to modulate cellular function. A molecule can be targeted to a particular type of cell, either through direct binding to an epitope on the surface of the cell, or through a linker that recognizes both the multivalent molecule and a marker on the cell surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of co-pending application Ser.No. 16/233,773 filed Dec. 27, 2018, now U.S. Pat. No. 11,274,131, whichis a Continuation of co-pending application Ser. No. 12/738,774 filedJun. 28, 2010, now U.S. Pat. No. 10,202,430, which is a National Stageapplication under 35 U.S.C. § 371 claiming priority from PCT ApplicationNo. PCT/US2008/080304 having an International Filing Date of Oct. 17,2008, which claims benefit of priority from U.S. Provisional ApplicationSer. No. 60/999,403 filed Oct. 18, 2007. Applicants claim the benefitsof 35 U.S.C. § 120 as to the U.S. applications, and priority under 35U.S.C. § 119 as to the said PCT Application and the U.S. Provisionalapplication, and the entire disclosures of all applications areincorporated herein by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant no. CA096859awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

This document relates to materials and methods for using multivalentmolecules (e.g., antibodies) to modulate cellular function.

BACKGROUND

Dendritic cells (DC) are efficient antigen-presenting cells (APC). Thesecells express class I and class II major histocompatibility complex(MHC) peptide-presenting molecules on their cell surfaces, along with aseries of costimulatory molecules (Banchereau and Steinman (1998) Nature392:245-252). Naive T cells express receptors for these DC ligands.Following recognition of peptide-antigen presented in the context ofclass I or class II molecules, the structure of the T cell membrane isreorganized, bringing together the elements of the T cell receptor withother cell-surface molecules, including the co-receptors CD4 or CD8 andthe costimulatory receptors CD28 and CTLA-4 (Monks et al. (1998) Nature395:82-86; and Wulfing and Davis (1998) Science 282:2266-2269).Interactions within the newly formed macromolecular complexes determinethe outcome of inductive events transduced into T cells by DC.

DC reside in a variety of tissues and display distinct tissue-associatedphenotypes (Strunk etal. (1997) J. Exp. Med. 185:1131-1136; Caux et al.(1996) J. Exp. Med. 184:695-706; Wu et al. (1996) J. Exp. Med.184:903-911; and Vremec et al. (1992) J. Exp. Med. 176:47-58). Therelationships among the cell lineages of these different subsets ofcells are not firmly established. A large body of work has emergedfocusing on DC generated in vitro from bone marrow or blood precursors(Mayordomo et al. (1995) Nat. Med. 1:1297-1302; Nonacs et al. (1992) J.Exp. Med. 176:519-529; Steinman and Witmer (1978) Proc. Natl. Acad. Sci.USA 75:5132-5136; and Young and Steinman (1990) J. Exp. Med.171:1315-1332). The cells generated in vitro express high levels ofclass I antigens and the series of costimulatory ligands associated withendogenous DC (Fagnoni et al. (1995) Immunology 85:467-474; andBanchereau et al. (2000) Annu. Rev. Immunol. 18:767-811). Importantly,they are able to efficiently activate naive T cells, a function that isthe signature of the DC.

Decavalent IgM antibodies display measurable binding avidity toantigens, even though binding affinity may be low. The multivalentstructure of pentameric IgM provides the potential for cross-linkingcell surface targets, endowing the soluble antibodies with biologicalpotential not normally associated with immune function. One such IgMantibody has been shown to bind and cross-link B7-DC on the surface ofDC. This monoclonal antibody, which is referred to herein as B7-DC XAbbut also has been called sHIgM12, rHIgM12, and Lym12, was originallyisolated from a Waldenstrom's macroglobulinemia patient. As described inU.S. Pat. No. 7,052,694, U.S. Ser. No. 10/881,661, and U.S. Ser. No.10/983,104 (all of which are incorporated herein by reference in theirentirety), B7-DC XAb can, for example, activate DC, potentiate immuneresponses, modulate existing states of immune responsiveness, and treator inhibit development of allergic asthma.

SUMMARY

As described herein, IgM antibodies and other multivalent molecules canbe used to cluster and cap cell surface molecules on a variety of celltypes, resulting in intracellular signaling and modulation of thetargeted cells' functions. These methods takes advantage of certain IgMmolecules' ability to bind with very low affinity to sets of endogenousligands. Cell function can be modulated by targeting an IgM to aparticular cell type via (1) a typical antibody interaction with anantigen normally expressed on the targeted cell, (2) a transgenicmolecule expressed on the cell surface containing an epitope recognizedby the IgM, or (3) a linker construct (e.g., a peptide or an antibody)with the ability to bind to the IgM and to a cell surface proteinspecific to the given cell type. Once the IgM has been recruited to thecell surface, its low affinity interaction with other endogenous ligandscan result in receptor and cell surface molecule clustering, initiatingintracellular signaling and modulating cell functions.

In one aspect, this document features a method for targeting amultivalent molecule to a cell, comprising: (a) contacting the cell witha linker molecule, wherein the linker molecule includes (i) an aminoacid sequence comprising an epitope to which the multivalent moleculespecifically binds and (ii) an amino acid sequence that bindsspecifically to a marker on the outer surface of the cell; and (b)contacting the cell with the multivalent molecule. The multivalentmolecule can be an antibody (e.g., an IgM antibody). The linker moleculecan consist of a polypeptide. The linker molecule can be a chimericantibody.

In another aspect, this document features a method for targeting amultivalent molecule to a cell, comprising: (a) contacting the cell witha nucleic acid encoding a polypeptide, wherein the polypeptide includes(i) an amino acid sequence that directs the polypeptide to the cell'splasma membrane and (ii) an amino acid sequence comprising an epitope towhich the multivalent molecule specifically binds; (b) culturing thecell under conditions in which the polypeptide is expressed andlocalized to the plasma membrane such that the epitope is located on theexterior of the cell; and (c) contacting the cell with the multivalentmolecule. The multivalent molecule can be an antibody (e.g., an IgMantibody).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic showing IgM binding to a cell surface receptor.IgM antibodies can interact with cells via transgenic moleculesexpressed on the cell surface that contain an epitope recognized by theIgM (A), or a linker peptide (B) or antibody (C) having the ability tobind to the IgM and to a cell surface protein specific to the given celltype. Once recruited to the cell surface, the interaction of the IgMwith other endogenous ligands can result in receptor and cell surfacemolecule clustering (D).

FIGS. 2A to 2G are a series of pictures showing that a kinase activatedpathway regulates antibody-induced antigen uptake by matured DC. Maturedhuman monocyte-derived DC were treated with the MTAb B7-DC XAb forvarying time periods and evaluated for the phosphorylation status ofselect signaling pathway intermediates by immunoprecipitation with DAP12(MC457), Syk (4D10), and PLCy1 (MC490) reactive antibodies followed bywestern blot analysis using the phosphotyrosine reactive antibody 4G10as a probe (FIGS. 2A-2C). Samples of the cells blocked with indicatedconcentration of piceatannol (FIG. 2D) or with 10 mM of piceatannol(FIG. 2E) were analyzed for phosphorylation of Syk (FIG. 2D) and PLCγ1(FIG. 2E). To investigate the functional importance of these signalingintermediates in the regulation of antigen uptake by matured DCactivated with B7-DC XAb, varying concentrations of pharmacologicinhibitors were used to block the activity of p72Syk (piceatannol) andPLCγ1 (U73122) (FIGS. 2F and 2G, respectively). Antigen uptake wasassessed by pulsing cultures with FITC-conjugated ovalbumin andmeasuring intracellular fluorescence 16 hours later in CD11c positivecells by flow cytometry.

FIG. 3 is a plot showing that matured human monocyte-derived DC take upovalbumin when activated by the MTAb B7-DC XAb. Human DC were generatedfrom peripheral blood CD14⁺ monocytes by incubation in GM-CSF and IL-4for five days. Cultures were allowed to proceed untreated (immature DC)or were treated overnight with the TLR-3 agonist poly I:C (matured DC).On day six, the DC cultures were activated the isotype control antibodysHIgM39 or with the MTAb B7-DC XAb, as indicated. All cultures werepulsed with OVA-FITC at the time of treatment with IgM antibodies, andof OVA-FITC was assessed 24 hours later by flow cytometry.

FIGS. 4A and 4B are a series of plots showing the effects of kinaseinhibitors on OVA-FITC uptake by matured human DC, as determined by flowcytometry. For FIG. 4A, day 6 matured human monocyte-derived DC wereincubated in the presence or absence of the Src kinase inhibitor PP2 for30 minutes prior to activation with isotype control antibody sHIgM39 orthe MTAb B7-DC XAb, as indicated. All cells were pulsed with OVA-FITC atthe time of treatment with IgM antibody, and CD11c+ cells were analyzedfor OVA-FITC uptake 24 hours later by flow cytometry. For FIG. 4B, humanDC were pretreated with the indicated inhibitors, activated on day 6with the MTAb B7-DC XAb, pulsed with FITC-OVA, and analyzed the next dayfor uptake of FITC-OVA by flow cytometry Inhibitors used were 50 nM Bim1 (PKC inhibitor); 25 μM Y-27632 (Rho A inhibitor); 10 μM LY294002 (PI3kinase inhibitor); 10 μM U73122 (PLCy inhibitor); 10 μM PD98509 (MEKinhibitor); and 1 μM SB203580 (p38 MAP kinase inhibitor).

FIG. 5A to 5F is a series of histograms and blots indicating thattightly organized macromolecular caps assemble following B7-DC XAbtreatment. In FIG. 5A, the distribution of class II (labeled withAPC-conjugated antibody) and CD80/CD86 molecules (labeled withPE-conjugated antibodies) was visualized by FRET following treatment ofhuman monocyte derived DC with B7-DC XAb (open histograms) or withisotype control antibody (filled histograms). For FIG. 5B, mouse bonemarrow derived DC were labeled, treated, and analyzed as in FIG. 5A.FIG. 5C indicates the inability of the IA^(b) specific IgM antibody25-9-3 (open dark histograms), B7-DC XAb (open light histograms), andisotype control antibody treated samples (filled histograms) to induceFRET (top left and middle panels). Binding of the class II specific IgMantibody to the mouse DC analyzed is shown (top right panel). FIG. 5Dindicates the ability of B7-DC-specific IgG antibody (open histograms onleft of each panel) or no IgG antibody (open histograms on right of eachpanel) to inhibit FRET induced by the MTAb B7-DC XAb. Absence of FRETinduced by Isotype control antibody is shown in reference (filledhistograms). For FIGS. 5E and 5F, immunoprecipitates of membraneproteins from B7-DC XAb or control antibody treated DC were isolatedusing biotin tagged IA^(b)-specific KH74 antibody. The precipitates wereanalyzed by Western blot using mouse-specific TREM-2 and CD40 antibodiesas probes.

FIG. 6A to 6C is a series of histograms and pictures showing that TREM-2is recruited into the MTAb-induced cap on mouse DC and expression ofTREM-2 is important for MTAb-induced phosphorylation of the adapterDAP-12 and the protein kinase Syk. FIG. 6A: six day cultures of bonemarrow-derived mouse DC were differentiated in GM-CSF and IL-4 weretagged with antibodies specific for the class II molecule IA^(b)(25-9-17-APC) and for TREM-2 (237916-PE) for 15 minutes prior tostimulation with the MTAb B7-DC XAb (open histograms) or with isotypecontrol antibody sHIgM39 (filled histograms). Cells were fixed in 0.1%paraformaldehyde prior to analysis by flow cytometry. FRET between PEand APC was assessed collecting light emitted by APC followingexcitation of PE with a 488 nm laser. Expression of TREM-2 on the cellswas assessed by staining with 237916-PE antibody alone (open histogram)or isotype control antibody (closed histogram, bottom-right panel). ForFIG. 6B, mouse bone marrow-derived DC were transduced on day 2 ofculture with a retrovirus expressing an hnRNA specific for TREM-2 or acontrol virus containing a scrambled sequence. On day 5 of culture, thetransduced cells were not treated (0′ time), activated with the isotypecontrol antibody sHIgM39, or activated with B7-DC XAb for 5 minutes.DAP12 was immunoprecipitated from cell lysates with antibody MC457,resolved by electrophoresis, and the blotted filters were probed withthe phosphotyrosine-specific antibody 4G10. FIG. 6C: the lysates used inthe experiments from FIG. 6B were immunoprecipitated with theSyk-specific antibody (4D10) and analyzed by western blot for thepresence of phosphotyrosine.

FIGS. 7A and 7B is a series of pictures and a pair of graphs indicatingthat distinct pathways are activated by B7-DC XAb. FIG. 7A is a pair ofpictures of western blot analysis for phosphorylation of DAP12 and Sykin B7-DC XAb or control antibody-treated mouse DC, as indicated. FIG. 7Bis a graph plotting percent CD11c+inguinal lymph node cells in miceafter OVA-FITC was introduced into the footpads of mice treatedsystemically with isotype control antibody or the MTAb B7-DC XAb(n=3/group). Cells were analyzed by flow cytometry 24 hours afterOVA-FITC introduction.

FIG. 8 is a graph plotting IL-6 levels in supernatants of cultured DC,showing that CD40 KO DC do not secrete IL-6 following activation withB7-DC XAb. Six day mouse bone marrow-derived DC from wild type or IL-6KO mice were stimulated with isotype control antibody sHIgM39 or B7-DCXAb. IL-6 levels in culture supernatants were assessed 48 hours later byELISA.

FIGS. 9A-9B, 9C and 9D are a series of graphs and pictures demonstratingthat MTAbs share activation mechanisms. In FIGS. 9A and 9B, freeintracellular calcium levels were assessed by flow cytometry in Indo-1labeled mouse DC (FIG. 9A) or human DC (FIG. 9B) following stimulationwith control antibody (dark) or B7-DC XAb (light histograms). The bottompanels in FIGS. 9A and 9B show calcium levels in DC preincubated withB7-DC specific IgG antibody and treated with B7-DC XAb (dark) or treatedwith ionomycin (light histogram). FIG. 9C shows a series of histogramsobtained using transfected Jurkat cells expressing the mouse class Igene K^(b)/L^(d) (top panels) or a chimeric class I gene containing the5A peptide mimetope recognized by the MTAb rHIgM22 (bottom panels) weretagged with antibodies specific for human CD4 (PE) and CD28 (APC) priorto treatment for 15 minutes with the control antibody sHIgM39, the MTAbrHIgM22, or the IgM antibody 28-13-3, specific for the K^(b)/L^(d)molecule. Cells were analyzed for FRET by flow cytometry. FIG. 9D is apicture of a western blot using whole cell lysates of Jurkat cells thatwere analyzed for global tyrosine phosphorylation by western blot.

FIG. 10 is a series of histograms showing recruitment of C28 and humanMHC class I molecules into a molecular cap on Jurkat cells expressing arHIgM22-reactive peptide mimetope. Human Jurkat cells were transientlytransfected with a mouse class I carrier gene (K^(b)/L^(d); top panels)or with a chimeric class I gene expressing an rHIgM22-reactive peptidemimetope (K^(b)/L^(d)-5A; middle panels). The transfected cells weretagged with a pan human class I-specific antibody (PE) and CD28-specificantibody (APC) 15 minutes prior to treatment with either the humanisotype control antibody sHIgM39, the MTAb rHIgM22, or the mouse IgMantibody 28-13-3, specific for the K^(b)/L^(d) carrier protein. Thecells were fixed 15 minutes after treatment with the IgM antibodies andanalyzed by flow cytometry for evidence of FRET between the PE and APCfluorochromes. Bottom panels: stable Jurkat transfectants expressingeither the K^(b)/L^(d) carrier protein (filled histograms) or themimetope tagged K^(b)/L^(d)-5A protein (open histograms), were analyzedfor FRET following treatment of cells pre-stained with pananti-HLA-A.B,C-PE and anti-CD4-APC with MTAb rHIgM22. The treated cellswere analyzed for FRET at the indicated time points.

FIGS. 11A and 11B depicts the amino acid sequences of B7-DC XAb. FIG.11A shows the variable (Vk) and constant (Ck5454) domains (SEQ ID NOS:4and 5, respectively) of the B7-DC XAb light chain. FIG. 11B shows thevariable (Vh) and constant (CH1, CH2, CH3, CH4) domains (SEQ ID NOS:6,7, 8, 9, and 10, respectively) of the sHIgM12 heavy chain.

FIG. 12 depicts the nucleic acid sequences of B7-DC XAb. Nucleic acidssequences encoding the Vk and Vh domains (SEQ ID NOS:11 and 12,respectively) B7-DC XAb.

FIG. 13 is a series of plots showing surface expression of Kb-peptideconstructs in Balb/c bone-marrow derived DCs, as detected by stainingwith antibody B8-24-3.

FIG. 14 is a series of histograms showing induction of FRET between CD80PE and Class II APC in B7DC−/− DCs expressing the Kb-peptide constructs.Wild type DCs or retrovirally transduced B7DC−/− were stained with CD80PE and Class II APC. Cells were treated with hIgM12 (red), hIgM22(blue), or 28-13-3 (green). FRET of CD80 PE with Class II APC wasmeasured by fluorescence in channel FL3.

FIG. 15 is a series of histograms showing restoration of antigenpersistence in CpG-treated DCs expressing Kb-peptide constructs upontreatment with the appropriate MTab. DCs were untreated (blue) ortreated with 10 ug/mL CpG (red) 48 hours prior to harvesting. They werethen pulsed with Ovalbumin-Alexa647 and either hIgM12, hIgM22, or28-13-3, and Ovalbumin-647 levels were measured in CD11c positive cellsby flow cytometry.

FIG. 16 is a series of plots showing conversion of OT-II CD4 Treg cellsto a Th17 phenotype by co-incubation with DCs expressing the Kb-peptideconstructs treated with MTabs. OT-II CD4+CD25+ splenic Tregs wereisolated by MACS and added 1:1 to DCs pulsed with Ovalbumin and treatedwith hIgM12, hIgM22, or 28-13-3. After 48 hours, the T cells wereharvested, stained with CD4, and internally stained with FoxP3 PE andIL-17 APC. Expression was assayed by flow cytometry, and cells weregated on CD4.

FIG. 17 is a graph depicting protection of mice from B16-Ova tumorchallenge by injection of Ova-pulsed DCs expressing the Kb-peptideconstructs treated with MTabs. Tumor area was measured daily, with tumorarea on day 11 represented in the graph.

DETAILED DESCRIPTION

Described herein are multivalent molecules that can activate thefunction of particular cell types. This work has implications fortreatment of a wide variety of human diseases. For example, monoclonalIgM therapeutic antibodies (MTAbs) have been identified that canactivate cells in the dendritic cell (DC) and oligodendrocyte lineages,inducing, e.g., immune modulation and remyelination of denuded axons(Nguyen et al. (2002) J. Exp. Med. 196:1393-1398; Warrington et al.(2000) Proc. Natl. Acad. Sci. U.S.A. 97:6820-6825; and Miller andRodriguez (1995) J. Immunol. 154:2460-2469). A remarkable feature ofMTAbs is their tendency to bind to and activate homologous cells inrodents and humans (Warrington et al., supra; Radhakrishnan et al.(2003) J. Immunol. 170:1830-1838; and Radhakrishnan et al. (2007) J.Immunol. 178:1426-1432), facilitating the use of animal models inpreclinical studies. These antibodies are present in the normal humanrepertoire, and can be identified in patients with monoclonalgammopathies.

MTAbs can function at very low concentrations, similar to what isobserved with conventional growth factors (Warrington et al. (2007) J.Neurosci. Res. 85:967-976). While the clinical application of growthfactors has been hampered by their short half life and difficulty ofdelivery, these obstacles can be overcome by MTAbs. IgM antibodies arenatural blood products and have half lives in humans of days. Inaddition, these molecules activate cells to perform inherent “luxury”functions. For example, MTAbs can activate DC to stimulate killing oftumors by CD8+ T cells (Radhakrishnan et al. (2004) Cancer Res.64:4965-4972; and Heckman et al. (2007) Eur. J. Immunol. 37:1827-1835),reprogram cellular immunity to block allergic airway inflammation(Radhakrishnan et al. (2004) J. Immunol. 173:1360-1365; andRadhakrishnan et al. (2005) J. Allergy Clin. Immunol. 116:668-674), oractivate oligodendrocytes to make new myelin wraps around axons(Warrington et al. (2000), supra; and Miller et al. (1994) J. Neurosci.14:6230-6238). MTAbs thus represent a new class of clinically usefulreagent which may have wide applicability.

The mechanism by which MTAbs activate targeted cells has remainedobscure, as cell surface molecules bound by these antibodies are notwell defined. As disclosed herein, however, mechanisms underlying DCactivation by the human MTAb B7-DC XAb have been characterized. Usingthese principles, it has been demonstrated that a second human MTAb,rHIgM22, an antibody that induces myelin repair in models of multiplesclerosis, can activate cells in the same fashion. Both therapeuticantibodies can rapidly induce formation of multimolecular caps on thesurface of targeted cells, recruit signaling molecules that are known tocontrol important cellular functions, and activate a series ofintracellular signals in response.

MTAbs can modulate immune responsiveness and oligodendrocyte maturationby targeting cells in situ and inducing preprogrammed cellularfunctions. MTAbs capable of inducing anti-tumor immunity, blockingallergic airway inflammation, and inducing remyelination of denudedaxons can employ a common cellular activation mechanism. For example,these antibodies can cross-link cell surface molecules and assemblemacromolecular signaling complexes by recruiting receptor and adaptermolecules into clusters, thereby activating key signaling pathways.Additional MTAbs that target cells specifically throughout the body canbe identified within the normal human antibody repertoire, providing thebasis for the development of novel therapeutic approaches to treatdisease.

As described herein, IgM antibodies or other multivalent molecules canbe used to cluster and cap cell surface molecules on a variety of celltypes, resulting in intracellular signaling and modulation of thetargeted cells' functions. These methods take advantage of certain IgMmolecules' ability to bind with very low affinity to sets of endogenousligands. In some embodiments, cell function can be modulated bytargeting the IgM to a particular cell type via a “typical” antibodyinteraction with an antigen normally expressed on the targeted cell, ora transgenic molecule expressed on the cell surface containing anepitope recognized by the IgM (e.g., as depicted in FIG. 1A). In somecases, an IgM antibody can be targeted to a particular cell type via alinker molecule. A linker can interact with both an IgM via a mimetoperecognized the IgM, and with a cell surface molecule specific to thegiven cell type. A linker can be, for example, a specially designedpeptide or nucleic acid (FIG. 1B), or an antibody linked to a mimetoperecognized by IgM (FIG. 1C). Once the IgM has been recruited to the cellsurface, its low affinity interaction with other endogenous ligands canresult in clustering/capping of the receptors, cell surface molecules,and adapter proteins (FIG. 1D), initiating intracellular signaling andmodulating cell functions.

This ability to target a “generic” MTAb to a variety of different celltypes using a linker can have a number of advantages. For example,because every IgM does not have sufficient affinity to bind effectivelyto endogenous ligands, a single MTAb can be used against a variety ofdifferent cell types. This can be especially useful if an IgM isidentified that has no affinity for a specific receptor or a particularcell type. In addition, other types of multivalent molecules (e.g.,tetramers, specially designed beads, and other constructs) can bedesigned to bind a specific linker and to have low affinity forendogenous ligands found on all cell types.

Linker polypeptides can be designed using any suitable means, includingphage display. Such methods can allow the design of linkers specific tocell types where a cell-type specific receptor/surface molecule has notbeen identified. Moreover, linker polypeptides can be designed usingvery small peptides, such that the full construct is about 15 to about40 amino acids in length (e.g., about 20 amino acids, about 25 aminoacids, about 30 amino acids, about 35 amino acids, or about 40 aminoacids in length). Such linkers can be synthesized using standardtechniques.

Further, the ability to target MTAbs to different cell types using alinker system can have enormous therapeutic potential. For example, alinker can be used to target an antibody specifically to a T_(reg) cellor to a stem cell, enhancing or inhibiting its activation fortherapeutic purposes. For example, a stem cell could be activated usingan MTAb and a linker to promote differentiation either in vivo or exvivo, providing a means for creating more differentiated stem cells fora variety of treatments (e.g., cardiac stem cells for treatingmyocardial infarction, or pancreatic islet cells for treating diabetes).

In addition, the ability to use a linker can be combined with other usesof therapeutic antibodies, such that the antibodies have multipleeffects. For example, a linker peptide could be used to make the “B7-DCXAb” antibody described herein simultaneously therapeutic againstdendritic cells through its ability to bind B7-DC and T_(reg) cells (orany other cell type) through the linker.

Polypeptides and Antibodies

The molecules provided herein typically are polypeptides, and antibodiescan be particularly useful (see below), but other multivalent moleculesthat can bind and cross-link molecules on the surface of cells also canfunction in this capacity. Examples of such molecules include, withoutlimitation, multivalent RNA or DNA aptamers.

As used herein, a polypeptide is an amino acid chain, regardless oflength or post-translational modification (e.g., phosphorylation orglycosylation). A polypeptide can contain an amino acid sequence that issimilar to the amino sequence of B7-DC AXb, for example. A polypeptidecan contain, e.g., an amino acid sequence that is at least 80.0%identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%,99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to99.9% identical) to the amino acid sequence set forth in SEQ ID NO 4 orSEQ ID NO:6. In some embodiments, a polypeptide can further contain anamino acid sequence that is at least 80.0% identical (e.g., 80.0%,85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95% to 99.9%, from96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to theamino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, or SEQ ID NO:10. Percent sequence identity is calculated bydetermining the number of matched positions in aligned nucleic acidsequences, dividing the number of matched positions by the total numberof aligned nucleotides, and multiplying by 100. A matched positionrefers to a position in which identical nucleotides occur at the sameposition in aligned nucleic acid sequences. Percent sequence identityalso can be determined for any amino acid sequence.

To determine percent sequence identity, a target nucleic acid or aminoacid sequence is compared to the identified nucleic acid or amino acidsequence using the BLAST 2 Sequences (B12seq) program from thestand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained from Fish & Richardson's web site (World Wide Web atfr.com/blast) or the U.S. government's National Center for BiotechnologyInformation web site (World Wide Web at ncbi.nlm.nih.gov). Instructionsexplaining how to use the B12seq program can be found in the readme fileaccompanying BLASTZ.

B12seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: −iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); −j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); −p is set toblastn; −o is set to any desired file name (e.g., C:\output.txt); −q isset to −1; −r is set to 2; and all other options are left at theirdefault setting. For example, the following command can be used togenerate an output file containing a comparison between two sequences:C:\B12seq c:\seq1.txt −j c:\seq2.txt −p blastn −o c:\output.txt −q −1−r2. To compare two amino acid sequences, the options of B12seq are set asfollows: −i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); −j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); −p is setto blastp; −o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq c:\seq1.txt −jc:\seq2.txt −p blastp −o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence (e.g., SEQ ID NO:4), or by anarticulated length (e.g., 100 consecutive nucleotides or amino acidresidues from a sequence set forth in an identified sequence), followedby multiplying the resulting value by 100. For example, a nucleic acidsequence that has 98 matches when aligned with the sequence set forth inSEQ ID NO:4 is 92.5 percent identical to the sequence set forth in SEQID NO:4 (i.e., 98/106*100=92.5). It is noted that the percent sequenceidentity value is rounded to the nearest tenth. For example, 75.11,75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16,75.17, 75.18, and 75.19 is rounded up to 75.2. It is also noted that thelength value will always be an integer.

The amino acid sequences of the polypeptides provided herein can havesubstitutions, deletions, or additions with respect to the amino acidsequences set forth in SEQ ID NOS:4 and 6. A polypeptide having an aminoacid sequence that is modified (e.g., by substitution) with respect toSEQ ID NO:4 and/or SEQ ID NO:6 can have substantially the same orimproved qualities as compared to a polypeptide containing the aminoacid sequence identical to that set forth in SEQ ID NO:4 and SEQ IDNO:6. A substitution can be a conserved substitution. As used herein, a“conserved substitution” is a substitution of an amino acid with anotheramino acid having a similar side chain. A conserved substitutiontypically can be a substitution with an amino acid that makes thesmallest change possible in the charge of the amino acid or size of theside chain of the amino acid (alternatively, in the size, charge or kindof chemical group within the side chain) such that the overall peptideessentially retains its spatial conformation but has altered biologicalactivity. Examples of conserved changes include, without limitation, Aspto Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu, andSer to Cys, Thr or Gly. Alanine is commonly used to substitute for otheramino acids. The 20 essential amino acids can be grouped as follows:alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophanand methionine having nonpolar side chains; glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine having uncharged polar sidechains; aspartate and glutamate having acidic side chains; and lysine,arginine, and histidine having basic side chains (see, e.g., Stryer,Biochemistry (2^(nd) edition) W. H. Freeman and Co. San Francisco(1981), pp. 14-15; and Lehninger, Biochemistry (2^(nd) edition, 1975),pp. 73-75). Conservative substitutions can include substitutions madewithin these groups.

Molecules provided herein can be antibodies. The terms “antibody” and“antibodies” encompass intact molecules as well as fragments thereofthat can bind to a particular antigen. Antibodies can be polyclonalantibodies, monoclonal antibodies, humanized or chimeric antibodies,single chain Fv antibody fragments, Fab fragments, and F(ab)₂ fragments.Polyclonal antibodies are heterogeneous populations of antibodymolecules that are specific for a particular antigen, while monoclonalantibodies are homogeneous populations of antibodies to a particularepitope contained within an antigen.

An antibody can be of any immunoglobulin (Ig) class, including IgM, IgA,IgD, IgE, and IgG, and any subclass thereof. Antibodies of the IgM class(e.g., B7-DC XAb) typically are pentavalent and can be particularlyuseful. Immune complexes containing Ig molecules that are cross-linked(e.g., cross-linked IgG) and are thus multivalent also can beparticularly useful.

As used herein, an “epitope” is a portion of an antigenic molecule towhich an antibody binds. Antigens can present more than one epitope atthe same time. For polypeptide antigens, an epitope typically is aboutfour to six amino acids in length, and can include modified (e.g.,phosphorylated or glycosylated) amino acids. Two differentimmunoglobulins can have the same epitope specificity if they bind tothe same epitope or set of epitopes.

Polyclonal antibodies are contained in the sera of immunized animals.Monoclonal antibodies can be prepared using, for example, standardhybridoma technology. In particular, monoclonal antibodies can beobtained by any technique that provides for the production of antibodymolecules by continuous cell lines in culture as described, for example,by Kohler et al. (1975) Nature 256:495-497, the human B-cell hybridomatechnique of Kosbor et al. (1983) Immunology Today 4:72, and Cote et al.(1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and the EBV-hybridomatechnique of Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. pp. 77-96 (1983). A hybridoma producing monoclonalantibodies can be cultivated in vitro or in vivo.

Antibodies also can be isolated from, for example, the serum of anindividual. The B7-DC XAb antibody, for example, was isolated from humanserum as described in U.S. Pat. No. 7,052,694. Suitable methods forisolation include purification from mammalian serum using techniquesthat include, for example, chromatography.

Antibodies also can be produced by, for example, immunizing host animals(e.g., rabbits, chickens, mice, guinea pigs, or rats) with an immunogen(e.g., an antigen or epitope). An immunogen can be producedrecombinantly, by chemical synthesis, or by purification of the nativeprotein, and then used to immunize animals by injection of thepolypeptide. Adjuvants can be used to increase the immunologicalresponse, depending on the host species. Suitable adjuvants includeFreund's adjuvant (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin (KLH), and dinitrophenol. Standard techniques can be used toisolate antibodies generated in response to the immunogen from the seraof the host animals.

Antibodies such as B7-DC XAb also can be produced recombinantly. Theamino acid sequence (e.g., the partial amino acid sequence) of anantibody can be determined by standard techniques, and a cDNA encodingthe antibody or a portion of the antibody can be isolated from the serumof the subject (e.g., the human patient or the immunized host animal)from which the antibody was originally isolated. The cDNA can be clonedinto an expression vector using standard techniques. The expressionvector then can be transfected into an appropriate host cell (e.g., aChinese hamster ovary cell, a COS cell, or a hybridoma cell), and theantibody can be expressed and purified. See, for example, U.S. Ser. No.10/983,104. Antibody fragments that have specific binding affinity foran antigen also can be generated by techniques such as those disclosedabove. Such antibody fragments include, but are not limited to, F(ab′)2fragments that can be produced by pepsin digestion of an antibodymolecule, and Fab fragments that can be generated by reducing thedisulfide bridges of F(ab′)2 fragments. Alternatively, Fab expressionlibraries can be constructed. See, for example, Huse et al. (1989)Science 246:1275-1281. Single chain Fv antibody fragments are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge (e.g., 15 to 18 amino acids), resulting in a singlechain polypeptide. Single chain Fv antibody fragments can be producedthrough standard techniques, such as those disclosed in U.S. Pat. No.4,946,778. Such fragments can be rendered multivalent by, for example,biotinylation and cross-linking, thus generating antibody fragments thatcan cross-link a plurality of B7-DC polypeptides.

Nucleic Acids, Vectors, and Host Cells

Nucleic acids encoding polypeptides and antibodies also are providedherein.

The term “nucleic acid” refers herein to both RNA and DNA, includingcDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Anucleic acid molecule can be double-stranded or single-stranded (i.e., asense or an antisense single strand). Nucleic acids include, forexample, cDNAs encoding antibody light and heavy chains.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acid molecules that normally flank one or both sidesof the nucleic acid in the genome in which it is normally found. Theterm “isolated” as used herein with respect to nucleic acids alsoincludes any non-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in its naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, aDNA molecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not considered an isolated nucleic acid.

The isolated nucleic acids disclosed herein can encode polypeptides asdescribed herein. For example, an isolated nucleic acid can encode apolypeptide containing an amino acid sequence that is similar oridentical to an amino acid sequence found in the variable or constantregions of B7-DC XAb (e.g., SEQ ID NOS:4, 5, 6, 7, 8, 9, 10, 11, and 12,shown in FIGS. 11A, 11B, and 12). In some embodiments, a nucleic acidcan encode a polypeptide containing an amino acid sequence that is atleast 80.0% identical (e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%,98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8, 99.9%, from 95% to 99.9%, from 96% to 99.9%, from 97% to 99.9%, orfrom 98% to 99.9% identical) to the amino acid sequence set forth in SEQID NO:4 or SEQ ID NO:6. The encoded polypeptide can further contain anamino acid sequence that is at least 80.0% identical (e.g., 80.0%,85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95% to 99.9%, from96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9% identical) to theamino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, or SEQ ID NO:10. In some cases, an isolated nucleic acidcan contain a nucleotide sequence that is at least 80.0% identical(e.g., 80.0%, 85.0%, 90.0%, 95.0%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, from 95%to 99.9%, from 96% to 99.9%, from 97% to 99.9%, or from 98% to 99.9%identical) to the nucleotide sequence set forth in SEQ ID NO:11 or SEQID NO:12. The method for determining percent sequence identity isprovided above.

Isolated nucleic acid molecules can be produced using standardtechniques, including, without limitation, common molecular cloning andchemical nucleic acid synthesis techniques. For example, polymerasechain reaction (PCR) techniques can be used to obtain an isolatednucleic acid molecule encoding an antibody. Isolated nucleic acids alsocan be chemically synthesized, either as a single nucleic acid molecule(e.g., using automated DNA synthesis in the 3′ to 5′ direction usingphosphoramidite technology) or as a series of polynucleotides. Forexample, one or more pairs of long polynucleotides (e.g., >100nucleotides) can be synthesized that contain the desired sequence, witheach pair containing a short segment of complementarity (e.g., about 15nucleotides) such that a duplex is formed when the polynucleotide pairis annealed. DNA polymerase is used to extend the polynucleotides,resulting in a single, double-stranded nucleic acid molecule perpolynucleotide pair.

Vectors containing nucleic acids such as those described herein also areprovided. A “vector” is a replicon, such as a plasmid, phage, or cosmid,into which another DNA segment may be inserted so as to bring about thereplication of the inserted segment. An “expression vector” is a vectorthat includes one or more expression control sequences, and an“expression control sequence” is a DNA sequence that controls andregulates the transcription and/or translation of another DNA sequence.

In the expression vectors provided herein, a nucleic acid (e.g., anucleic acid encoding the light and/or heavy chains of an antibody suchas B7-DC XAb) can be operably linked to one or more expression controlsequences. As used herein, “operably linked” means incorporated into agenetic construct so that expression control sequences effectivelycontrol expression of a coding sequence of interest. Examples ofexpression control sequences include promoters, enhancers, andtranscription terminating regions. A promoter is an expression controlsequence composed of a region of a DNA molecule, typically within 100 to500 nucleotides upstream of the point at which transcription starts(generally near the initiation site for RNA polymerase II). To bring acoding sequence under the control of a promoter, it is necessary toposition the translation initiation site of the translational readingframe of the polypeptide between one and about fifty nucleotidesdownstream of the promoter. Enhancers provide expression specificity interms of time, location, and level. Unlike promoters, enhancers canfunction when located at various distances from the transcription site.An enhancer also can be located downstream from the transcriptioninitiation site. A coding sequence is “operably linked” and “under thecontrol” of expression control sequences in a cell when RNA polymeraseis able to transcribe the coding sequence into mRNA, which then can betranslated into the protein encoded by the coding sequence. Expressionvectors thus can be useful to produce antibodies as well as othermultivalent molecules.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerousvectors and expression systems are commercially available from suchcorporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.),Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

Host cells containing vectors also are provided. The term “host cell” isintended to include prokaryotic and eukaryotic cells into which arecombinant expression vector can be introduced. As used herein,“transformed” and “transfected” encompass the introduction of a nucleicacid molecule (e.g., a vector) into a cell by one of a number oftechniques. Although not limited to a particular technique, a number ofthese techniques are well established within the art. Prokaryotic cellscan be transformed with nucleic acids by, for example, electroporationor calcium chloride mediated transformation. Nucleic acids can betransfected into mammalian cells by techniques including, for example,calcium phosphate co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, or microinjection. Suitable methods fortransforming and transfecting host cells are found in Sambrook et al.,Molecular Cloning: A Laboratory Manual (2^(nd) edition), Cold SpringHarbor Laboratory, New York (1989), and reagents for transformationand/or transfection are commercially available (e.g., LIPOFECTIN®(Invitrogen); FUGENE® (Roche, Indianapolis, Ind.); and SUPERFECT®(Qiagen, Valencia, Calif.)).

Also provided herein are cells (e.g., DC) contacted in vitro with amultivalent polypeptide (e.g., an IgM antibody) as described herein.

Compositions

The molecules described herein (e.g., antibodies such as B7-DC XAb andnucleic acids encoding linkers or transgenic receptors) can beincorporated into compositions, as can isolated cells that have beencontacted with one or more molecules as described herein. Compositionsprovided herein also can contain a molecule (e.g., a polypeptide) thatis immobilized on a solid substrate, such as a bead. The compositionscan be administered to a subject in order to modulate cellular function(e.g., to enhance DC function and potentiate an immune response).

Methods for formulating and subsequently administering therapeuticcompositions are well known to those skilled in the art. Dosagestypically are dependent on the responsiveness of the subject to themolecule, with the course of treatment lasting from several days toseveral months, or until a suitable immune response is achieved. Personsof ordinary skill in the art routinely determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages can vary dependingon the relative potency of an antibody, and generally can be estimatedbased on the EC₅₀ found to be effective in in vitro and/or in vivoanimal models. Dosage typically is from 0.01 μg to 100 g per kg of bodyweight (e.g., from 1 μg to 100 mg, from 10 μg to 10 mg, or from 50 μg to500 μg per kg of body weight). Compositions containing the moleculesprovided herein may be given once or more daily, weekly, monthly, oreven less often.

In addition to the molecules provided herein, the compositions describedherein further can contain antigens that will elicit a specific immuneresponse. Suitable antigens include, for example, polypeptides orfragments of polypeptides expressed by tumors and pathogenic organisms.Killed viruses and bacteria, in addition to components of killed virusesand bacteria, also are useful antigens. Such antigens can stimulateimmune responses against tumors or pathogens.

The molecules (e.g., antibodies, other polypeptides, or nucleic acids)can be admixed, encapsulated, conjugated or otherwise associated withother molecules, molecular structures, or mixtures of compounds such as,for example, liposomes, receptor targeted molecules, or oral, topical orother formulations for assisting in uptake, distribution and/orabsorption.

In some embodiments, a composition can contain a molecule providedherein in combination with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are pharmaceutically acceptablesolvents, suspending agents, or any other pharmacologically inertvehicles for delivering antibodies to a subject. Pharmaceuticallyacceptable carriers can be liquid or solid, and can be selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, and other pertinent transport and chemicalproperties, when combined with one or more therapeutic compounds and anyother components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, without limitation: water;saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions containing molecules provided herein can beadministered by a number of methods, depending upon whether local orsystemic treatment is desired. Administration can be, for example,parenteral (e.g., by subcutaneous, intrathecal, intraventricular,intramuscular, or intraperitoneal injection, or by intravenous (i.v.)drip); oral; topical (e.g., transdermal, sublingual, ophthalmic, orintranasal); or pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols). Administration can be rapid (e.g., by injection)or can occur over a period of time (e.g., by slow infusion oradministration of slow release formulations). For administration to thecentral nervous system, antibodies can be injected or infused into thecerebrospinal fluid, typically with one or more agents capable ofpromoting penetration across the blood-brain barrier.

Compositions and formulations for parenteral, intrathecal orintraventricular administration include sterile aqueous solutions (e.g.,sterile physiological saline), which also can contain buffers, diluentsand other suitable additives (e.g., penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers).

Compositions and formulations for oral administration include, forexample, powders or granules, suspensions or solutions in water ornon-aqueous media, capsules, sachets, or tablets. Such compositions alsocan incorporate thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders.

Formulations for topical administration include, for example, sterileand non-sterile aqueous solutions, non-aqueous solutions in commonsolvents such as alcohols, or solutions in liquid or solid oil bases.Such solutions also can contain buffers, diluents and other suitableadditives. Pharmaceutical compositions and formulations for topicaladministration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be useful.

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsion formulations are particularlyuseful for oral delivery of therapeutic compositions due to their easeof formulation and efficacy of solubilization, absorption, andbioavailability. Liposomes can be particularly useful due to theirspecificity and the duration of action they offer from the standpoint ofdrug delivery.

Molecules featured herein can encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to a subject, is capable of providing (directly orindirectly) the biologically active metabolite or residue thereof.Accordingly, for example, this document provides pharmaceuticallyacceptable salts of molecules such as antibodies, prodrugs andpharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. A prodrug is a therapeutic agent that is prepared in aninactive form and is converted to an active form (i.e., drug) within thebody or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. The term “pharmaceutically acceptablesalts” refers to physiologically and pharmaceutically acceptable saltsof the antibodies useful in methods provided herein (i.e., salts thatretain the desired biological activity of the parent antibodies withoutimparting undesired toxicological effects). Examples of pharmaceuticallyacceptable salts include, but are not limited to, salts formed withcations (e.g., sodium, potassium, calcium, or polyamines such asspermine); acid addition salts formed with inorganic acids (e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, ornitric acid); salts formed with organic acids (e.g., acetic acid, citricacid, oxalic acid, palmitic acid, or fumaric acid); and salts formedwith elemental anions (e.g., bromine, iodine, or chlorine).

Compositions additionally can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions also can include compatible, pharmaceutically activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms of the compositionsdescribed herein, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents, and stabilizers.Furthermore, the composition can be mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,penetration enhancers, and aromatic substances. When added, however,such materials should not unduly interfere with the biologicalactivities of the other components within the compositions.

Pharmaceutical formulations as disclosed herein, which can be presentedconveniently in unit dosage form, can be prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients (i.e., the antibodies) with the desired pharmaceuticalcarrier(s). Typically, the formulations can be prepared by uniformly andintimately bringing the active ingredients into association with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product. Formulations can be sterilized ifdesired, provided that the method of sterilization does not interferewith the effectiveness of the molecules(s) contained in the formulation.

Methods

This document provides methods for targeting multivalent molecules to aparticular cell type. Such methods can include, for example, contactinga cell with a multivalent polypeptide (e.g., an IgM antibody) that canbind specifically to a particular epitope. In some embodiments, theepitope can be present on the surface of the cell. In such cases, theepitope can be contained within a native polypeptide that is expressedon the cell surface, or can be a mimetope contained within a transgenicpolypeptide that is expressed on the cell surface. In the latter case,the method can include contacting the cell with a nucleic acid encodingthe transgenic polypeptide, such that the transgenic polypeptide isexpressed on the cell surface. In some embodiments, the epitope can beincluded in a linker molecule (e.g., a polypeptide or antibody) thatinteracts with both the multivalent molecule and with a cell surfacemolecule. In such cases, the method can include contacting the cell withthe linker or with a nucleic acid encoding the linker.

The methods provided herein can be used to modulate the function(s) ofthe cells to which the multivalent molecules and other components areadministered. In some embodiments, for example, a multivalent molecule(e.g., an antibody such as B7-DC XAb) or a composition containing themultivalent molecule or a nucleic acid encoding the molecule can beadministered to a mammal (e.g., a dog, a cat, a horse, a cow, a rabbit,a rat, a mouse, or a human). As described above, the molecule(s) orcomposition can be administered using any suitable systemic or localmethod. Systemic methods of administration include, without limitation,oral, topical, or parenteral administration, as well as administrationby injection. Local methods of administration include, for example,direct injection into a tumor.

Methods also are provided that include contacting an isolated cell(e.g., a dendritic cell, or any other type of cell) in vitro with amultivalent polypeptide (e.g., an IgM antibody) as described above, andadministering the cell to a subject.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Materials and Methods

Mice: C57BL/6J, B6.129s4-CD80−/−CD86−/−, CNCr.129P2-Cd40tm1Kik/J, andB6.12952-IL6tmlKopf/J mice, 6-8 weeks old, were obtained from JacksonLaboratories (Bar Harbor, Me.) and used for generation of bone marrowderived DC. Class II knock out mice (L. Madsen et al. (1999) Proc. Natl.Acad. Sci. USA 96:10338-10343) were a gift from Dr. Chella David, MayoClinic. TREM-2 knock out mice (Turnbull et al. (2006) J. Immunol.177:3520-3524), bred in the mouse colony at Washington University Schoolof Medicine, St. Louis, Mo., were provided by Dr. Marco Colonna.Pregnant rats were purchased from Harlan Sprague (Indianapolis, Ind.).All animals were maintained at the Mayo Clinic animal facility for atleast one week prior to use.

Reagents: Appropriate fluorophore labeled antibodies against murineI-A^(b) (25-9-17), murine class II specific IgM (25-9-3), APC labeledanti-mouse CD11c (HL3) FITC labeled anti-human class II (TU39), FITClabeled anti-human CD28 (CD28.2), APC labeled anti-human CD28 (CD28.2),PE labeled anti-human CD4 (RPA-T4) and PE labeled anti-human HLA A, B, C(G46-2.6) were purchased from BD PharMingen (San Jose, Calif.).Appropriate fluorophore labeled antibodies against mouse class II(M5/114.15.2), CD80 (16.10A1), CD86 (GL-1), CD11c (N418), CD40 (IC 10),APC labeled antibody against human DR (LN3), PE labeled anti-human CD80(2D10.4), CD86 (IT2.2), murine B7-DC specific IgG antibody (TY25), andhuman B7-DC IgG antibody (MIH18) were purchased from eBioscience (SanDiego, Calif.). All secondary appropriately fluorophore labeled F(ab)²fragment antibodies were obtained from Jackson Immunoresearch(Westgrove, Pa.). An IgM antibody (28-13-3) specific for mouse class IH-2 K^(b) was obtained from a hybridoma cell line from ATCC(HB-41)(Manassas, Va.). Antibodies against the C terminal portion of NF-κB(sc372) and the protein kinase Syk (4D10) were obtained from Santa CruzBiotechnology (Santa Cruz, Calif.). DAPI and DNAse were obtained fromSigma Aldrich (St. Louis, Mo.). Anti phosphotyrosine, 4G10 and goatanti-mouse antibody were obtained from Upstate Cell Signaling Solutions(Lake Placid, N.Y.). Anti-mouse TREM-2 antibodies (237920) for flowcytometry and (237916) for western blot analysis were purchased from R&Dsystems (Minneapolis, Minn.). Rabbit antibodies against PLC yl (MC490),and DAP12 (MC457) were developed by Dr. Paul Leibson, Mayo Clinic.Ovalbumin labeled with FITC or APC was purchased from Molecular Probes(Eugene, Oreg.). Protein A Sepharose was purchased from PierceBiotechnology (Rockford, Ill.). All inhibitors were obtained fromCalbiochem (San Diego, Calif.) unless otherwise indicated. Piceatenolwas obtained from Sigma Aldrich. Rac-1 inhibitor, NSC23766 was a giftfrom Dr. Daniel Billadeau, Mayo Clinic. LPS was obtained from SigmaAldrich. CpG oligonucleotides (Radhakrishnan et al. (2005) Proc. Natl.Acad. Sci. U.S.A. 102:11438-11443) were synthesized at the Mayo corefacility. The polynucleotide pI:C was purchased from Calbiochem (SanDiego, Calif.). All MTABs were purified as described (Warrington et al.(2000), supra; and Radhakrishnan et al. (2003) J. Immunol.170:1830-1838) and used at 10 μg/ml.

Generation of DC: DC from mouse bone marrow were generated as previouslydescribed (Inaba et al. (1992) J. Exp. Med. 176:1693-1702). Briefly,bone marrow was isolated from the long bones of the hind legs.Erythrocytes were lysed by treatment with ammonium chloride/potassiumbicarbonate/EDTA at 37° C. The remaining cells were plated 1×10⁶cells/ml in six-well plates (BD Biosciences, San Jose, Calif.) in RPMIcontaining 10 ng/ml of murine GM-CSF and 1 ng/ml of murine IL-4(PeproTech, Rocky Hill, N.J.). The cells were incubated at 37° C. with5% CO2. After 48 hours, the cells were washed and replated with RPMIcontaining the same concentration of GM-CSF and IL-4 for another 5 days.Human DC were derived from CD14+ mononuclear cells isolated fromperipheral blood using magnetic bead sorting (Miltenyi Biotec, Auburn,Calif.). Briefly, a buffy coat was obtained from a unit of blood donatedby a normal human donor. Peripheral blood mononuclear cells (PBMC) wereisolated by centrifugation over Ficoll-Paque PLUS (Amersham Biosciences,Piscataway, N.J.) and the CD14+ cells were separated by positivemagnetic cell sorting. The isolated cells were incubated in RPMI 1640supplemented with 5% human AB serum (HP 10220, Valley Biomedical,Winchester, Va.), 1% sodium pyruvate (Mediatech, Herndon, Va.), 1%non-essential amino acids (Mediatech), 1 ng/ml IL-4 (R&D Systems,Minneapolis, Minn.) and 50 ng/ml GM-CSF (Berlex, Richmond, Calif.) at1×10⁶ cells/ml, 3 ml/well in six well plates for 6-8 days at 37° C. with5% CO₂. Maturation of DC was achieved by addition of TLR ligands for aperiod of 24 hours before being used for antigen uptake assays.

Generation of mixed glial cultures: Oligodendrocytes from rat pups wereobtained as per the protocol previously described (Warrington et al.(2000), supra). Briefly, tissue culture plates coated with poly-D-lysine(25 ug/mL) prepared in water for 1-2 hours at 37° C. were used forculturing the cells. Rat pup brains were removed under sterileconditions. Cerebral hemispheres, hindbrain, cerebella, thalamus,hippocampus, and meninges were removed and minced into 1 mm chunks witha sterile single-edge razor blade. Tissue chunks were trypsinized,subjected to shaking for 30-40 minutes at 37° C., and heat inactivatedfetal calf serum was added to a final concentration of 10% to inactivatethe trypsin. DNAse at 1:50 was added to the solution and incubated for5-10 minutes. This process was repeated until a single cell suspensionwas achieved. The cell suspension was layered over a cushion of 4% BSAand then centrifuged. Supernatant was collected and the cells wereresuspended in DMEM containing 10% FCS at a density of 20×10⁷cells/plate. Media was changed 4 days post-seeding and every 3 daysthereafter. The plates were shaken at day 9 to obtain a heterogenouspopulation of oligodendrocytes.

Production of shTREM2 and shControl virus: Oligos containing the shTREM2sequence (5′-TGATGCTGGAGATCTCTGGGTTCAAGAGACCCAGAGATCTCCAGCATCTTTTTTC-3;SEQ ID NO:1) and shControl sequence(5′-TGACTGCTGAAGGTCGCTTGTTTCAAGAGACCAAGCGACCTCCAGCATCTTTTTTC-3′; SEQ IDNO:2) (Warrington et al. (2000), supra) were synthesized and cloned intothe pSUPER RNAi System (provided by Dr. Daniel Billadeau, Mayo Clinic)using the key restriction sites Bgl II and Hind III. The sequence wasconfirmed by automated sequencing of the vectors. The resulting vectorswere co-transfected with VSV-G and gagpol plasmids (both provided by Dr.Richard Vile, Mayo Clinic) into 293T cells. Supernatant was collected at48 and 72 hours, pooled, filtered through a 0.45 micron filter andfrozen until used for transduction.

Transduction of DC: For transducing DC with the virus, 1 ml ofsupernatant containing the scrambled virus or shDNA containing dominantnegative TREM-2 virus was mixed with 2 ml of RPMI. Cytokines were addedto a final concentration of 10 ng/ml murine GM-CSF and 1 ng/ml murineIL-4 at day 2 of DC culture. Cells were maintained for another 3 daysbefore using the DC for antigen uptake assay as mentioned above or foranalysis of phosphorylation status of DAP12 and Syk proteins.

Immunoblots: In experiments involving assessment of the phosphorylationstatus of various protein kinases using whole cell lysate, dendriticcells of mouse or human origin or Jurkat cells were stimulated at theindicated times with control antibody or B7-DC XAb, and were lysed onice for 10 minutes in 1 ml buffer containing 20 mM Tris-HCl, 40 mM NaCl,5 mM EDTA, 50 mM NaF, 30 mM Na₄P₂O₇, 0.1% BSA, 1 mM Na₃VO₄, 1 mM PMSF, 5μg/ml aprotinin, 10 μg/ml leupeptin, and 1% Triton X-100. Cellulardebris was removed by centrifugation at 20,800×g for 5 minutes at 4° C.and the supernatant was used in SDS-PAGE analysis for phosphorylatedtyrosine proteins. For immunoprecipitation, antibody against mouse Syk(4D10) or PLC γ1 (MC490) or DAP12 (MC457) at 10 μg per sample was boundto protein A-Sepharose beads at 4° C. for 2 hours under constantrotation. Supernatants from cell lysate after stimulation were incubatedwith antibody coupled beads for 2 hours at 4° C. with constant rotation.In experiments involving inhibition of Syk kinase, cells were incubatedwith 10 μM of Piceatenol for 30 minutes before being stimulated withcontrol antibody or B7-DC XAb. For suppression of TREM-2, DC weretransduced as described above and were stimulated with control antibodyor B7-DC XAb on day six, lysed, and subjected to immunoprecipitation.Protein complexes were eluted in 40 μl of SDS sample buffer, resolved bySDS-PAGE, and transferred to Immobilon-P membranes (Millipore).Tyrosine-phosphorylated proteins were detected using theanti-phosphotyrosine specific antibody, 4G10, followed by goatanti-mouse IgG coupled to Horse Radish Peroxidase (Santa CruzBiotechnology) and the SuperSignal detection system (PierceBiotechnology, Rockford, Ill.). Thereafter, total protein was visualizedby staining the membrane with Ponceau staining solution (PierceBiotechnology) for 30 seconds for analysis of whole cell lysate. Forimmunoprecipitation assays, the membrane was stripped with 7M guanidine,blocked with BSA, and probed with the antibody against the whole proteinfollowed by protein A coupled to HRP (Amersham Biosciences) and theSuperSignal detection system. For pull down assays from the macromolecular complex, affinity purified antibody against mouse Class II(I-A^(b)) (KH74) was used for immunoprecipitation. The supernatants wereresolved by SDS-PAGE, probed with anti-mouse TREM-2 antibody (237920)and detected by Goat-anti mouse antibody coupled to HRP, or probed withaffinity purified anti-mouse CD40 (1C10) and detected by Goat-anti mouseantibody coupled to HRP.

NFκB activation assay: DC of murine or human origin were stimulated with10 μg/ml of control antibody or the MTAb B7-DC XAb for 30 minutes, whilestimulation of oligodendrocytes was achieved by addition of 10 μg/ml ofMTAb sHIgM39, 04, or sHIgM22 for same period of time. All groups werefixed and permeabilized using a Cytofix/Cytoperm Kit (BD PharMingen, SanDiego, Calif.) for 20 minutes on ice. Subsequently, rabbit antibodyagainst a C terminal peptide of NF-κB (sc372) was added, incubated for30 minutes at 4° C. and washed three times with Cytoperm Buffer.Anti-rabbit FITC was used to detect the bound sc372 antibody byincubating the cells for 30 minutes (green). Cells were washed threetimes before the nuclei were stained with DAPI (blue) (Sigma Aldrich).The cells were then harvested from the plate, cytospin slides were made,and cells were visualized using a LSM510 Laser scanning confocalmicroscope (Carl-Zeiss Inc, Oberkochen, Germany) at 40.times.magnification.

IL-6 ELISA: DC of wild type origin or from CD40 deficient mice werestimulated with 10 μg/ml control antibody or B7-DC XAb for 48 hours.Supernatants from the different treatment groups were harvested andquantified for the amount of IL-6 by sandwich ELISA as per themanufacturer's protocol (eBioscience, San Diego, Calif.).

Live cell imaging for visualization of macromolecular complex: DC ofmurine origin were stained with anti-Class II-FITC (MF/114.15.2), andeither anti-CD80-PE (16.10A1)/CD86-PE (GL-1), or anti-CD11c-PE (N418).DC of human origin were stained with anti-Class II-FITC (LN3) andanti-CD80-PE (2D10.4)/CD86-PE (IT2.2). All incubations were carried outfor 15 minutes at 37° C. The cells were subsequently stimulated with 10μg/ml of control (sHIgM39) or B7-DC XAb, and were observed every 5minutes using time lapse confocal imaging at 40.times. magnificationwith a LSM510 Laser scanning confocal microscope having a 37° C. stage(Carl-Zeiss Inc, Oberkochen, Germany). Jurkat cells were pre stainedwith anti-human CD28-FITC (CD28.2) for 15 minutes, followed by additionof 10 μg/ml of MTAb control antibody sHIgM39, MTAb sHIgM22, oranti-K^(b) IgM (28-13-3), and were and analyzed in the same manner asthe DC.

Flow Cytometry and FRET: The flow cytometry approach for FRET was usedas a way of quantifying molecular aggregation on the cell surface andwas carried out as described previously (Block et al. (2001) J. Immunol.167:821-826). Briefly, DC of murine origin were stained with anti-ClassII APC (M5/114.15.2) and anti-CD80-PE (16.10A1)/CD86-PE(GL-1) for 15minutes to visualize FRET between these molecules. Monitoring for FRETinduced by interaction between TREM-2 and Class-II molecules wasachieved by incubating the cells with the abovementioned anti-Class-IIantibody and anti-TREM-2-PE (237920). DC of human origin were stainedfor 15 minutes with APC-anti class II (LN3) and anti-CD80-PE(2D10.4)/CD86-PE (IT2.2). In experiments involving blocking of B7-DC,both fluorophore labeled antibodies and purified anti-mouse B7-DC(TY-25) or purified anti-human B7-DC (MIH18) IgG monoclonal antibody wasadded at 10 μg/ml for 15 minutes. Jurkat cells were stained withanti-human Class I-PE (G46-2.6) or anti-human CD4 (RPA-T4) andanti-human CD28-APC (CD28.2) for 15 minutes. Cells were stimulated withcontrol antibody or B7-DC XAb or purified anti-mouse class II IgM(25-9-3) (experiments involving DC) or sHIgM22 (experiments involvingJurkat cells) and aliquots from different groups were taken at differenttime points. After 15 minutes of incubation, the cells were washed andfixed in 2% paraformaldehyde prior to analysis by FACS using aFACSCALIBER™ (BD Biosciences, Franklin Lakes, N.J.). Data collected aslog₁₀ fluorescence were analyzed using CELLQUEST™ (BD Biosciences). TheFRET signal was visualized in FL3 channel (650-670 nm LP).

Antigen uptake assay: Antigen uptake experiments were carried out asdescribed previously (Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci.U.S.A. 102:11438-11443). Day 5 DC were matured with TLR ligand CpG-ODNfor mouse DC, or with pI:C for human DC. The matured cells wereincubated with ovalbumin labeled with FITC or APC and control antibodyor B7-DC XAb for two hours, washed and analyzed by FACSCALIBUR™ (BectonDickinson). In studies involving inhibitors, cells were pretreated 30minutes as indicated or at 10 μM concentration prior to addition ofcontrol antibody or B7-DC XAb. In vivo antigen uptake assays involvingwild type or TREM-2−/− mice were carried out by intravenous injection of10 μg of control antibody or B7-DC XAb on three consecutive days (days−1, 0 and +1). Ovalbumin coupled to FITC was injected in the right footpad at 1 mg/ml in PBS at 100 μA volume. The draining lymph node cellswere harvested 48 hours after injection of antigen, and stained withanti-mouse CD11c-PE. The data are expressed as percent green cells(ovalbumin-FITC) that are positive for CD11c.

Calcium flux assays: Changes in the levels of intracellular Ca²⁺ wereassessed in Indol-loaded cells by flow cytometry as described previously(Takahashi et al. (2005) J. Exp. Med. 201:647-657). Briefly, 5×10⁶ DC/mlwere incubated with 5 μM Indo-1 (Calbiochem) at 37° C. for 30 minutes.The cells were washed twice in serum free media and resuspended at10^(6/)ml in PBS containing 0.5% BSA. The samples were analyzed by flowcytometry using a UV laser. Violet (390 nm) and blue (500 nm)fluorescence emissions were recorded. Baseline was recorded for twominutes. Antibody was added to the cell suspension (10 μg/ml of controlantibody or B7-DC XAb) and the measurements were recorded for 8additional minutes. In blocking experiments, mouse or human DC werepre-incubated with 10 μg/ml of purified anti-mouse B7-DC antibody(TY-25) or purified anti-human B7-DC antibody (MIH18) for 15 minutes,followed by collection of data for calcium flux as described above.Ionomycin added at 1 μg/ml after 2 minutes served as a positive control.The blue to violet ratio was calculated using FlowJo software (Tristar,Ashland, Oreg.).

Phage Display Library: A disulfide constrained heptapeptide phagedisplay library from New England Biolabs (Ipswich, Mass.) was used forphage display (Felici et al. (1991) J. Mol. Biol. 222:301-310). Therandomized sequence in the library is flanked by cysteine residues,allowing disulfide cross-link that results in phage display of cyclizedpeptides. Host strain ER2738 and phage titering were followed per NewEngland Biolab's instruction manual. Briefly, a tissue culture dish wascoated with 30 μg/ml of sHIgM22 in 2 ml of PBS overnight at 4° C. Thefollowing day, an exponential culture of ER2738 was grown inTetracycline LB media. After blocking and washing, the phage library wasdiluted to 2×10¹¹ in 1 ml of 1×TBS containing Tween-20 buffer. Uponremoval of unbound phage by repeated washing, bound phage was elutedwith 1.5 ml of 0.2M Glycine pH 2.2. After neutralizing with 1M Tris pH9.0, eluted phage was added to an exponentially growing culture of ER2738 bacteria and was allowed to amplify for 4.5 hours at 37° C. PEG wasused to precipitate the culture supernatant. After 4 rounds, theamplified phage was cloned by limiting dilution. Twenty single-phagecontaining colonies were picked for sequence analysis. A peptidesequence (5A) present in 7 of 20 colonies was identified.

Generation of K^(b)/L^(d)-5A class I-peptide chimera: The 5A consensuspeptide (PPWQSWI; SEQ ID NO:3) coding sequence was introduced into theK^(b)/L^(d) gene (Pullen et al. (1989) J. Immunol. 143:1674-1679) bysite directed mutagenesis (Stratagene, Cedar Creek, Tex.) as permanufacturer instructions. Briefly, two complementary oligonucleotideswere generated. The 5′ strand included a sequence encoding the consensus5A peptide (PPWQSWI; SEQ ID NO:3) flanked by a HSAC (SEQ ID NO:13)spacer on the 5′ end and 20 bases of the K^(b) intron sequence. The 3′end included a CG spacer followed by 20 bases of the alpha one domain ofthe class I gene. Oligonucleotides were isolated by PAGE purification. Amixture consisting of 290 ng of each oligo with 500 ng of 5A7-K^(b)template DNA was amplified with Ultra HF polymerase for 18 cycles at 95°C. for 1 minute and 55° C. for 1 minute, followed by one cycle ofelongation at 68° C. for 10 minutes. After treatment with theendonuclease DpnI, the samples were transformed into XL-10 competentcells and plated on Ampicillin plates, and 12 randomly selected colonieswere sequenced with ABI's 3730XL capillary sequencer. Positive colonieswere grown up using Qiagen's Endotoxin Free Mega plasmid purificationkit.

Transfection of Jurkat T cells: The day prior to transfection, plasmidDNA of each construct was purified by ethanol precipitation. Jurkatcells (15×10⁶ cells) were resuspended in 250 μl RPMI, and 50 μl ofresuspended DNA was added. After 10 minutes of incubation at roomtemperature, the whole suspension was transferred to a #640 BTX 4 mmGapped cuvette and electroporation was carried out at 315 volts, 1pulse, 10 pulse length, and at low voltage using a BTX Model 820electroporator. Cells (1×10⁶/ml) were resuspended in RPMI containing 10%FCS. After overnight incubation at 37° C., cells were harvested and usedfor assays.

Example 2—Effects of B7-DC XAb Binding

Human MTAb B7-DC XAb binds to the costimulatory molecule B7-DC (PD-L2)on the surface of DC and in a B7-DC-dependent manner activates DCfunctions (Nguyen et al., supra; Radhakrishnan et al. (2003) J. Immunol.170:1830-1838; and Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci.U.S.A. 102:11438-11443). To determine the mechanism of cellularactivation for MTAbs, intracellular events rapidly triggered by B7-DCXAb binding were examined. As shown in FIG. 2, phosphorylation ofcellular proteins was induced as early as one minute after treatment ofhuman DC with B7-DC XAb. As no signaling domains are evident in thestructures of mouse or human B7-DC (Tseng et al. (2001) J. Exp. Med.193:839-846), association of B7-DC with molecules containing signalingdomains may be required for this activation. The adaptor molecule DAP12can couple receptors lacking innate signaling capability to downstreamsignaling pathways (Tomasello et al. (2000) Immunity 13:355-364; Bouchonet al. (2001) J. Exp. Med. 194:1111-1122; and Snyder et al. (2004)Immunol. 173:3725-3731), and was rapidly phosphorylated uponcross-linking B7-DC on human DC (FIG. 2A). In other systems, Src familykinases are linked to Syk family kinases by DAP12 (Obergfell et al.(2002) J. Cell Biol. 157:265-275). Analysis of immunoprecipitated Syk,before and after treatment of human DC with B7-DC XAb, revealedphosphorylation of the protein tyrosine kinase (FIG. 2B) concomitantwith the phosphorylation of PLCγ1 (FIG. 2C), a frequent downstreamsubstrate for Syk (Obergfell et al., supra).

Similar to the previously observed response by matured mouse DC(Radhakrishnan et al. (2005) Proc. Natl. Acad. Sci. U.S.A.102:11438-11443), matured human DC responded to B7-DC XAb treatment byregaining the ability to take up antigen (FIG. 3). To address theimportance of the signaling intermediates generated by cross linkingB7-DC in the regulation of antigen uptake in mature DC, pharmacologicinhibitors of Src, Syk, and PLCy were used to block steps in theactivation pathway. As shown in FIG. 4A, blockade of Src kinases withPP2 resulted in inhibition of antigen uptake by matured DC in responseto B7-DC cross-linking Pretreatment of DC with piceatannol inhibitedphosphorylation of Syk and PLCγ 1 (FIGS. 2D and 2E), and also inhibiteduptake of tagged proteins by matured DC in a dose responsive manner(FIG. 2F), as did inhibition of PLCγ activation using the inhibitorU73122, but not when the cells were treated with the inactive analogueU73343 (FIG. 2G). Taken together, these findings indicate that the MTAbB7-DC XAb activates a Src.fwdarw.PLCγ1 pathway that is required foractivation of antigen uptake in matured DC induced by B7-DC XAbtreatment. Using similar modes of pharmacological inhibition, it wasobserved that calcium dependent PKC activity, PI3 kinase, and the Rhofamily GTPase RAC1 are important factors regulating antigen uptake bymatured DC activated with cross-linking B7-DC (FIG. 4B). In contrast,neither Rho-A nor MAP family kinases p38 appear to influence thisfunction (FIG. 4B).

To address how B7-DC might be coupled to these downstream signalingpathways, the possibility that binding B7-DC with B7-DC XAb mightrecruit other membrane proteins into a complex on the membrane wasevaluated. DC cell-surface membrane molecules, including MHC class II,CD80, and CD86, were tagged with fluoresceinated antibodies prior toactivation with cross-linking antibody. The distribution of the antibodytags reorganized into a distinctly capped cluster on the cell membranesof both mouse and human DC soon after treatment with B7-DC XAb, but notafter treatment with the irrelevant IgM isotype control antibodysHIgM39. To establish a quantitative and dynamic assessment of capformation, recruitment of molecules into the cap was measured usingresonance energy transfer (FRET), as described previously (Block et al.(2001) J. Immunol. 167:821-826). Within ten minutes of B7-DCcross-linking with the MTAb B7-DC XAb, class II, CD80, and CD86molecules moved into close juxtaposition on the DC membrane, asvisualized by a strong FRET signal among the fluorescently taggedmolecules (FIGS. 5A and 5B). The molecules remained close enough forFRET during the next 20 minutes of observation, indicating that a stablemacromolecular complex was formed.

An antibody of IgM isotype that binds to class II I-A^(b) moleculesexpressed by C57BL/6 mice failed to induce any co-localization of CD80and CD86 on mouse DC (FIG. 5C), underscoring the specialized capabilityof B7-DC XAb to induce these marked membrane rearrangements. The abilityof B7-DC XAb to induce the cap was abolished by pretreating human ormouse DC with B7-DC-specific IgG antibodies (FIG. 5D). This observationwas consistent with previous findings that the B7-DC XAb-inducedfunctional changes in DC are dependent on B7-DC (Radhakrishnan et al.(2003) J. Immunol. 170:1830-1838; and Radhakrishnan et al. (2005) Proc.Natl. Acad. Sci. U.S.A. 102:11438-11443). The importance of CD80, CD86,and class II molecules expressed on DC to B7-DC XAb-induced capformation was investigated using DC derived from CD80/CD86 double knockout and MHC class II knock out mice. Following treatment with B7-DC XAb,co-capping of CD80 and CD86 or CD11c with class II molecules stilloccurred, indicating that these particular molecules are not requiredfor cap formation.

TREM-2, a pattern recognition receptor expressed on monocytes andcultured DC, is known to activate DAP12 (Obergfell et al., supra; andDaws et al. (2001) Eur. J. Immunol. 31:783-791). As visualized by FRET,an association of TREM-2 with class II molecules was observed within 5minutes, and increased by 15 minutes after treatment of DC with B7-DCXAb (FIG. 6A). This association of TREM-2 with class II molecules wasconfirmed by co-immunoprecipitation of TREM-2 with the class II moleculeIA^(b) in lysates isolated from DC 5 minutes after B7-DC XAb treatment(FIG. 5E). In contrast, TREM-2 was not associated with class IImolecules on DC following treatment with isotype control antibody inmock activation.

To evaluate the functional importance of TREM-2 in DC activation withthe MTAb B7-DC XAb, antigen uptake by matured, TREM-2 deficient DC wasassessed using an RNA knockdown strategy. A retrovirus containing adominant negative shDNA for TREM-2 was transduced into mouse bonemarrow-derived DC, substantially reducing the expression of TREM-2 onthe cell surface. This reduction was associated with the absence ofphosphorylation of DAP12 and Syk (FIGS. 6B and 6C). Furthermore, usingmatured DC, the shDNA-transduced cells were not induced to take upovalbumin when treated with the MTAb B7-DC XAb. When DC were transducedwith virus containing a scrambled shDNA sequence, expression of TREM-2,phosphorylation of DAP12 and Syk (FIGS. 6B and 6C), and enhancedovalbumin uptake proceeded in the same manner described previously withwild type DC not infected with retrovirus (Radhakrishnan et al. (2005)Proc. Natl. Acad. Sci. U.S.A. 102:11438-11443).

The important contribution of TREM-2 in the transduction of B7-DC XAbinduced signals was confirmed using DC derived from TREM-2 KO bonemarrow. While phosphorylation of DAP12 and Syk was readily induced inwild type DC activated with the MTAb B7-DC XAb, phosphorylation of thesesame signaling intermediates was not induced in TREM-2 KO mice (FIG.7A), and the matured KO DC did not regain the ability to take upovalbumin after antibody treatment. Together, these findings indicatethat B7-DC cross-linking leads to recruitment of TREM-2 to themacromolecular cap, and activation of DAP12. DAP12 is to recruit Syk, akinase functionally linked to PLCγ1, which is an upstream regulator ofantigen uptake in matured DC activated by B7-DC XAb. These findingsusing mouse DC, coupled with those described in FIG. 2 and FIG. 5 usinghuman cells, underscore the mechanistic parallels governing DCactivation in these two species by the MTAb B7-DC XAb.

To determine whether the role of TREM-2 in mediating signals induced byB7-DC XAb in DC generated in vitro also applies to cells in vivo,experiments were conducted to compare the ability of CD11c+ draininglymph node cells in wild type and TREM-2 KO mice treated systemicallywith B7-DC XAb or control antibody to take up labeled proteinsintroduced subcutaneously after antibody treatment. Whereas B7-DC XAbtreatment significantly activated uptake of labeled ovalbumin in thelymph nodes of wild type mice, the antibody had no effect on uptake ofthis antigen by CD11c+ cells in the lymph nodes of TREM-2 KO mice (FIG.7B). These findings indicate that the importance of TREM-2 in thesignaling pathway described for DC generated in vitro from bone marrowprecursors also applies to the response of natural DC in vivo.

DC can be activated by B7-DC XAb in the absence of TREM-2, as shown bythe mobilization of NF-κB in MTAb treated TREM-2 KO cells. To determinewhat other receptors regulating NF-κB activation might also be recruitedto the signaling complex by B7-DC XAb, the role of CD40 was examined.These experiments revealed that this receptor also is recruited to theB7-DC XAb-induced cap as indicated by its co-precipitation with class IImolecules following activation of DC with cross-linking antibody, butnot following treatment with isotype control IgM antibody (FIG. 5F). Thefunctional importance of CD40 in B7-DC XAb-mediated DC activation isindicated by the absence of NF-κB activation and IL-6 secretion (FIG. 8)by bone marrow derived DC generated from CD40 KO mice, while DC fromwild type mice responded to the cross-linking antibody by activatingNF-κB and producing IL-6. DC from CD40 KO mice displayed enhancedantigen uptake in response to treatment with B7-DC XAb, indicating thatTREM-2 expression was required for up-regulation of antigen uptake inmatured DC following B7-DC cross-linking, while the presence of CD40 wasrequired for activation of NF-κB and secretion of IL-6.

The delineation of signaling pathways linking the binding of B7-DC XAbto the DC cell surface to downstream cellular functions provides a basisfor evaluating the mechanisms of activation employed by other MTAbs. Themonoclonal MTAbs 04 (Asakura et al. (1998) J. Neurosci. 18:7700-7708)and rHIgM22 (Warrington (2000), supra; and Mitsunaga et al. (2002) FASEBJ. 16:1325-1327) bind oligodendrocytes and induce remyelination ofdenuded axons when administered systemically to mice. Primaryoligodendrocytes and oligoglial cell lines mobilize a calcium responsewhen exposed to the MTAbs 04 and rHIgM22 (Pas Soldan et al. (2003) Mol.Cell. Neurosci. 22:12-24; and Howe et al. (2004) Neurobiol. Dis.15:120-131). Treatment of both mouse (FIG. 9A) and human DC (FIG. 9B)with the MTAb B7-DC XAb also resulted in prolonged calcium signals,which were blocked when the DC were first treated with B7-DC specificIgG antibodies. Furthermore, CG4 cells are protected from apoptoticsignals enhancing their longevity under stressed conditions (Howe etal., supra), a phenotype also observed in culture when DC were treatedwith the MTAb B7-DC XAb (Nguyen et al., supra). Another parallel amongthese cells treated with MTAbs is the mobilization of activated NF-κB.These strong parallels observed among cells activated with the MTAbsB7-DC XAb, 04, and rHIgM22 suggest a common mechanism of membranerearrangement that leads to recruitment of signaling molecules intomacromolecular caps, mobilizing specific tissue responses in vitro andvivo. The precise nature of the molecules mediating MTAb induced signalsin oligodendrocytes remains undefined. Several different IgM antibodiesshare the ability to activate oligodendrocytes (Warrington et al.(2000), supra; Miller and Rodriguez, supra; Asakura et al., supra; PasSoldan et al., supra; and Howe et al., supra). These antibodies appearto target more than one cell surface molecule, as indicated by blockinga cellular activation of one IgM by its IgG switch variant, but theinability of that IgG antibody to block cellular activation by other IgMantibodies that target the same cells (Howe et al., supra).

One hypothesis is that the MTAbs are targeted to cells by moderatespecific binding affinity for molecules distinctly expressed by specificcell types. Once bound to these cell restricted epitopes, the antibodiesmay interact by secondary, weak cross-reactivity with other cell surfacemolecules that are moving on the membrane, drawing them into a tightcluster. The structure of the induced clusters may lead to activation ofcell surface molecules with intrinsic signaling capability, initiatingpreprogrammed changes in cellular function.

To test this hypothesis, a molecule displaying low but measurablebinding for the oligodendrocyte-specific MTAb rHIgM22 was engineered andintroduced into Jurkat cells to serve as bait and attract the MTAb tothe surface of a cell not normally targeted by the IgM antibody. Toconstruct this antibody target, a phage display peptide library wasscreened to identify a peptide mimetope that binds weakly to theremyelinating antibody sHIgM22. The peptide sequence was incorporatedinto the coding sequence for the N-terminus of a K^(b)/L^(d) chimericmouse class I molecule (Pullen et al. (1989) J. Immunol. 143:1674-1679)and the gene was expressed in human Jurkat T cells by transienttransfection. Previous studies had demonstrated that peptides introducedat the amino terminus of a mouse class I extracellular domain can beexpressed without disrupting the expression and structural integrity ofthe class I molecule (Hedley et al. (1989) J. Immunol. 143:1026-1031).Treatment of the transfected Jurkat cells with the MTAb sHIgM22 induceda molecular cap on the cell surface, and promoted a FRET signal betweenthe B7-family receptor CD28 and the class II co-receptor CD4 (FIG. 9D)and with a set of human class I molecules (FIG. 10). The ability of thesHIgM22 antibody to promote assembly of a macromolecular complex onJurkat cells also was confirmed using stable transfectants (FIG. 10).Enhanced tyrosine phosphorylation of Jurkat cellular proteins also wasdetected after treating cells transfected with the peptide-class Ichimeric genes with the MTAb rHIgM22, but not following treatment withan IgM antibody that binds to the carrier K^(b)/L^(d) mouse class Iheavy chain that carries the peptide mimetope. In cells transfected withthe parental class I carrier gene (not encoding the peptide mimetope),none of the IgM antibodies induced caps, FRET signals, orphosphorylation of cellular proteins (FIG. 9D). Thus, by introducing anartificial target capable of attracting rHIgM22 onto the surface ofJurkat cells, a signaling complex was assembled that resulted inactivation of cellular tyrosine kinases. This experimental systemvalidates the model, and demonstrates that two different MTAbs functionby recruiting cell surface proteins to a macromolecular complex.

The concept that antibodies can modulate cell functions by their abilityto bind cell surface receptors is well established (Sege and Peterson(1978) Proc. Natl. Acad. Sci. USA 75:2443-2447; and Taub and Greene(1992) Biochem. 31:7431-7435). In addition to specific engagement ofreceptors by antibodies raised by direct immunization, observedinteractions between naturally-occurring autoreactive antibodies andcells have given rise to the notion that natural antibodies may functionas ligands supporting normal development and homeostatic maintenance oftissue physiology (Ando et al. (1994) J. Biol. Chem. 269:19394-19398;Coutinho and Avrameas (1992) Scand. J. Immunol. 36:527-532; and Avrameas(1991) Immunol. Today 12:154-159). Until now, however, there has beenlittle mechanistic insight into how antibodies might function in thisway. IgM antibodies with their decavalent binding structure would seemto be natural cross-linkers, however, many studies using IgM antibodieshave required secondary cross-linkers to assemble molecular caps. Themolecules described herein represent a subset of naturally occurring IgMautoreactive antibodies that are highly somatically mutated,distinguishing them in some respect from traditional natural antibodies(Miller and Rodriguez (1995), supra; Asakura et al., supra; Mitsunaga etal., supra; and Van Keulen et al. (2006) Clin. Exp. Immunol.143:314-321), that have the capacity to induce macromolecular caps andactivate signaling pathways without secondary cross-linking.

The accumulating evidence leads to the prediction that IgM antibodiescapable of specifically activating a wide variety of cells in the humanbody can be identified. Recently, an IgM antibody has been describedthat activates neurons in culture, promoting neurite outgrowth(Warrington et al. (2004) J. Neuropathol. Exp. Neurol. 63:461-473). TheIgM antibodies already described from mice and human patients havetended to bind to equivalent cell types in rodents and humans(Warrington et al. (2000), supra; Radhakrishnan et al. (2003) J.Immunol. 170:1830-1838; and Radhakrishnan et al. (2007) J. Immunol.178:1426-1432), indicating that the recognized epitopes areevolutionarily conserved structures. These shared products are likelythe products of enzymatic modification of surface molecules. Thiscross-species reactivity demonstrates the potential clinical applicationof MTAbs using animal models of multiple sclerosis, cancer, and asthma(Warrington et al. (2000), supra; Radhakrishnan et al. (2004) CancerRes. 64:4965-4972; Radhakrishnan et al. (2004) J. Immunol.173:1360-1365; Miller et al. (1997) J. Neuroimmunol. 75:204-209; andBieber et al. (2002) Glia 37:241-249). An important feature of the humanMTAbs is that they were identified in patients with high titermonoclonal gammopathies. While each of these patients has high titers ofIgM antibody in their serum, none of them have developedantibody-associated pathologies causing abnormal kidney, liver,cardiovascular, or neurologic functions. By identifying MTAbs frommonoclonal gammopathy patients who are free of these antibody-associatedpathologies, one can select IgM antibodies for further development thatmay behave well as therapeutic agents when administered to patients.Once MTAbs are identified in patient serum and tested for therapeuticactivity using animal models of disease, recombinant sources of antibodycan be generated using reverse genetics, as described previously(Mitsunaga et al., supra; and Van Keulen et al., supra), and prepared asGMP-grade reagents for clinical study.

Example 3—A Surrogate Target System for Investigation of Human MTAbs

A phage display system (Ph.D.-C7C Peptide Library Kit, New EnglandBiolabs) was used to select 7-mer peptides in a cysteine loop for eitherhIgM12 or hIgM22. Briefly, a random peptide bacteriophage library waspanned against immobilized antibody. Unbound bacteriophage was washedaway, and bound bacteriophage was eluted, regrown and re-panned againstthe antibody. Multiple (three to seven) rounds of panning led todevelopment of a peptide consensus sequence for each antibody. Twosimilar consensus sequences were identified for hIgM22, while oneconsensus sequence was identified for hIgM12 (Table 1).

TABLE 1 Amino acid sequences of consensus peptide targets Name SequenceNo. of clones SEQ ID NO: hIgM22 peptides 22p3 CPSEHQWIC  4x 14 22p5aCPPWQSWIC  7x 15 hIgM12 peptides 12p4 CARNSTPPC  1x 16 12p9a CHQTEKLTC11x 17

Peptides 22p3 and 12p9A, which represented 4 and 11 clones,respectively, were selected. Each peptide was joined to the N-terminalsequence of the mouse MHC I molecule Kb with an Asp-Ser-Ala linker,permitting surface expression of the peptide. Kb was chosen due to itshigh level surface expression, previous work showing that peptides couldbe attached to Kb's N terminus without disrupting its structure, andpossession of an anti-Kb IgM (28-13-3). In addition, a Kb construct withboth peptides was constructed, with 22p3 N-terminal to 12p9A.

Kb, Kb-12p9A, Kb-22p3, and Kb-22p12p were placed in a VSV-G pseudotypedpBabe Puro retroviral vector, a vector based on the Maloney MurineRetrovirus. Previous experiments had shown that a high rate oftransduction can be achieved, as measured by GFP expression, and thatinfection with the retrovirus does not alter the gross biology of DCs.Retrovirus was produced by transient transduction of 293T cells, andsupernatant was concentrated 100:1 by ultracentrifugation on a 10%sucrose cushion. The retrovirus was titered with a colony forming unitassay of HT-1080 cells transduced with limiting dilutions of thesupernatant concentrate, followed by selection with 2 ug/mL puromycin.To make DCs, bone marrow was harvested from Balb/c, C57BL6 mice orB7DC−/− mice on a C57BL6 background.

Bone marrow was placed in RPMI+10% Cosmic Calf Serum with 1 ng/mL IL-4and 10 ng/mL GM-CSF, and plated at 10⁶/mL in 24 well plates. On day 2post-plating, media was aspirated; fresh media was added with 0.5 MOIretrovirus and 4 ug/mL polybrene. Using this system, the peptideconstructs were expressed in cultured DCs (FIG. 13).

To directly compare the activity of the MTabs hIgM12 and hIgM22, aseries of well-defined biological outputs of treatment of wild type DCswith hIgM12 was used. FRET was used as an output for formation of themultimolecular cap seen upon binding of hIgM12 to wild type DCs. Theemission of phycoerythrin (PE) was used to excite APC, which normallyemits in the FL4 channel but under FRET emits in the FL3 channel.Experiments were conducted to test FRET of CD40-PE to Class II APC, andCD80-PE to Class II APC (FIG. 14). Either wild type C57BL6 DCs orB7DC−/− DCs transduced with either B7DC or the Kb constructs were used.DCs were stained with the fluorochrome-labeled antibodies, and thentreated for 15 minutes at 37° C. with 10 μg/mL of either hIgM12, hIgM22,or 28-13-3. Wild type DCs responded to hIgM12 but not hIgM22 or 28-13-3,and knockouts with restored B7DC expression responded as well. Knockoutstransduced only with Kb did not respond to any antibody, while theKb-peptide constructs restored activity of the target antibody, suchthat Kb-12p9A DCs responded to hIgM12, Kb-22p3 DCs responded to hIgM22,and Kb-22p12p DCs responded to both antibodies.

The ability of Kb-peptide expressing DCs to alter antigen processing wasthen tested. Untreated DCs take up fluorochrome-labeled protein, and thefluorochrome labeled protein persists over 24 hours, as measured by flowcytometry. DCs treated with a Toll receptor agonist (LPS, CpG, orPolyI:C) 24 hours prior to addition of protein lose their ability totake up and maintain this antigen. Addition of hIgM12, but not otherMTabs, has been shown to restore the ability of TLR agonist-treated DCsto take up and maintain antigen. The ability of B7DC−/− DCs transducedwith the Kb-peptide constructs was compared to wild type DCs orknockouts transduced with B7DC. As previously shown, antigen persistencein CpG-treated wild type DCs or B7DC−/− DCs could be restored tountreated levels by addition of hIgM12, but not hIgM22 or 28-13-3.Knockouts transduced with Kb did not respond to any antibody, whilethose transduced with the Kb-peptide constructs responded to theappropriate MTAb (FIG. 15).

Previous experiments demonstrated the ability of DCs treated with hIgM12to convert CD4+CD25+Treg cells to a Th17 phenotype in anantigen-specific manner. This was measured by loss of FoxP3 expressionand gain of IL-17 expression, corresponding to loss of a repressivephenotype and gain of an effector phenotype. Studies were conducted tocompare the ability of wild type DCs or B7DC−/− B7DC reconstituted DCsto B7DC−/− transduced with the Kb-peptide constructs to convert Tregs toTh17 polarized T cells. DCs were pulsed with 10 ug/mL ovalbumin andtreated with 10 ug/mL of either hIgM12, hIgM22, or 28-13-3, 24 hoursprior to addition of T cells. Splenic OT-II Tregs were isolated by MACsand added to DC cultures at a 1:1 ratio. After 48 hours ofco-incubation, T cells were harvested, stained with CD4 PerCP, andpermeabilized and stained with FoxP3 PE and IL-17 APC. Samples were runby flow cytometry and cells were gated by high CD4 expression.Conversion was measured by loss of FoxP3 expression and gain of IL-17expression. Wild type DCs and B7DC reconstituted knockouts treated withhIgM12 but not hIgM22 or 28-13-3 converted Tregs to Th17 polarized cells(FIG. 16). Knockouts transduced with Kb did not respond to any antibody,while those transduced with the Kb-peptide constructs responded only tothe appropriate antibody—hIgM12 when expressing Kb-12p9A and hIgM22 whenexpressing Kb-22p3.

DCs treated with hIgM12 can induce potent anti-tumor immunity. It hasbeen shown (e.g., as described in U.S. Pat. No. 7,052,694) that miceinjected intravenously with either hIgM12 directly or DCs pre-treatedwith hIgM12 and tumor antigen are protected against challenge by B16melanoma cells, while untreated or control IgM treated mice had to besacrificed due to disease progression. A variation of this model wasused to determine whether Kb-peptide expressing DCs could induceprotective tumor immunity. C57BL6 mice were injected subcutaneously inthe right flank with 5×10⁵ B16-Ova cells, a B16 variant engineered toexpress ovalbumin. Wild type DCs or B7DC−/− DCs transduced with mB7DC orthe Kb constructs were pulsed with 10 ug/mL ovalbumin and treated with10 ug/mL of either hIgM12 or hIgM22, 24 hours prior to injection. Beforeinjection, DCs were washed and resuspended in 200 μL HBSS. 10⁶ DCs wereinjected intravenously by tail vein concurrent to B16 injection. Threemice were used in each treatment group. Tumor area was measured daily,and mice were sacrificed when tumor area reached 289 mm². Mice treatedwith wild type DCs stimulated with hIgM12 or B7DC reconstituted knockoutmice were successfully protected from tumor outgrowth, while hIgM22failed to protect (FIG. 17). Mice treated with knockout DCs transducedwith Kb did not respond to either treatment. Mice injected with DCsexpressing Kb-12p9A and treated with hIgM12, or DCs expressing Kb-22p3and treated with hIgM22 were protected, while the reciprocal antibodytreatment failed to offer protection. Thus, DC expressing Kb-peptidefusions were able to induce protective tumor immunity.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for targeting a multivalent molecule toa cell, comprising: (a) contacting the cell with a linker molecule,wherein the linker molecule includes (i) an amino acid sequencecomprising an epitope to which the multivalent molecule specificallybinds and (ii) an amino acid sequence that binds specifically to amarker on the outer surface of the cell; and (b) contacting the cellwith the multivalent molecule.
 2. The method of claim 1, wherein themultivalent molecule is an antibody.
 3. The method of claim 2, whereinthe antibody is an IgM antibody.
 4. The method of claim 1, wherein thelinker molecule consists of a polypeptide.
 5. The method of claim 1,wherein the linker molecule is a chimeric antibody.
 6. A method fortargeting a multivalent molecule to a cell, comprising: (a) contactingthe cell with a nucleic acid encoding a polypeptide, wherein thepolypeptide includes (i) an amino acid sequence that directs thepolypeptide to the cell's plasma membrane and (ii) an amino acidsequence comprising an epitope to which the multivalent moleculespecifically binds; (b) culturing the cell under conditions in which thepolypeptide is expressed and localized to the plasma membrane such thatthe epitope is located on the exterior of the cell; and (c) contactingthe cell with the multivalent molecule.
 7. The method of claim 6,wherein the multivalent molecule is an antibody.
 8. The method of claim7, wherein the antibody is an IgM antibody.
 9. A chimeric polypeptidecomprising a first amino acid sequence and a second amino acid sequence,wherein the first amino acid sequence comprises a Kb amino acidsequence, and where the second amino acid sequence comprises an epitopeto which a multivalent molecule binds.
 10. The chimeric polypeptide ofclaim 9, wherein the multivalent molecule is an antibody.
 11. Thechimeric polypeptide of claim 10, wherein the antibody is an IgMantibody.
 12. The chimeric polypeptide of claim 11, wherein the IgMantibody is hIgM12 or hIgM22.
 13. The chimeric polypeptide of claim 9,wherein the second amino acid sequence comprises SEQ ID NO:14 or SEQ IDNO:17.